Summary

Human embryonic stem cells (hESCs) self-renew indefinitely and give rise to
derivatives of all three primary germ layers, yet little is known about the
signaling cascades that govern their pluripotent character. Because it plays a
prominent role in the early cell fate decisions of embryonic development, we
have examined the role of TGFβ superfamily signaling in hESCs. We found
that, in undifferentiated cells, the TGFβ/activin/nodal branch is
activated (through the signal transducer SMAD2/3) while the BMP/GDF branch
(SMAD1/5) is only active in isolated mitotic cells. Upon early
differentiation, SMAD2/3 signaling is decreased while SMAD1/5 signaling is
activated. We next tested the functional role of TGFβ/activin/nodal
signaling in hESCs and found that it is required for the maintenance of
markers of the undifferentiated state. We extend these findings to show that
SMAD2/3 activation is required downstream of WNT signaling, which we have
previously shown to be sufficient to maintain the undifferentiated state of
hESCs. Strikingly, we show that in ex vivo mouse blastocyst cultures, SMAD2/3
signaling is also required to maintain the inner cell mass (from which stem
cells are derived). These data reveal a crucial role for TGFβ signaling
in the earliest stages of cell fate determination and demonstrate an
interconnection between TGFβ and WNT signaling in these contexts.

Introduction

Embryonic stem cells are a population of multipotent, self-renewing cells
that are derived from the epiblast of mammalian blastocyst embryos and that
retain this developmental identity even after prolonged culture in vitro
(Rossant, 2001). ES cells can
be induced to differentiate to functional cell types of all three primary germ
layers in vitro, and upon integration into host animals, they have the
capacity to contribute to all cell types of the embryo, including the germline
(Smith, 2001). These qualities
have made ES cells valuable resources for the introduction of complex genetic
modifications into mice; and the recent isolation and culture of human
embryonic stem cells (hESCs) (Thomson, 1998) has drawn a great deal of
attention to their biology and the potential they offer for regenerative
medicine, as well as the study of early human development.

Little is known about the signaling pathways that govern the unique
properties of hESCs. Maintenance of mouse embryonic stem cell (mESC) identity
was initially found to be dependent on extrinsic factors that were produced by
a feeder layer of mouse embryonic fibroblasts (MEFs). Subsequently, it was
found that leukemia inhibitory factor (LIF) was produced by MEFs and was
sufficient to maintain mESC identity in the absence of a feeder layer. LIF,
however, is not the lone factor responsible for the maintenance of stem cell
identity in the mouse, as null mutants in which LIF/Stat3 signaling is
eliminated show no defect in the establishment of the stem cell compartment
(reviewed by Smith, 2001).
Furthermore, LIF is incapable of maintaining stem cell identity in hESCs
(Thomson et al., 1998;
Reubinoff et al., 2000;
Sato et al., 2004), suggesting
a contribution from other signaling pathways to the establishment and/or
maintenance of stem cell identity in mammalian development.

Models of early vertebrate development have described a role for multiple
signaling cascades in the emergence of pattern and cell identity
(Harland and Gerhart, 1997;
Gilbert, 2003). These models
have suggested a prominent role for TGFβ signaling in the earliest cell
fate decisions of embryogenesis, including neural induction and mesendoderm
specification in Xenopus (reviewed by
Munoz-Sanjuan and Brivanlou,
2002), and primitive streak and mesoderm formation in the mouse
(reviewed by Goumans et al., 2000). The TGFβ superfamily of ligands,
which contains ∼40 potential ligands in the human genome, signals through
two main branches: the SMAD1/5 branch, which transduces on behalf of BMP and
GDF ligands via the type I receptors ALK1, ALK2, ALK3 and ALK6 (ACVERL1,
ACVER1, BMPR1A and BMPR1B, respectively - Mouse Genome Informatics); and the
TGFβ/activin/nodal branch involves the activation of SMAD2/3 via ALK4,
and ALK5 and ALK7 (TGFBR1 and ACVR1C - Mouse Genome Informatics) (reviewed by
Shi and Massague, 2003). There
are also two inhibitory SMADs - SMAD6, which selectively inhibits SMAD1/5; and
SMAD7, which inhibits both branches of TGFβ signaling - that provide a
repressive input on the pathway. Upon activation by phosphorylation and
association with a common SMAD4, the receptor-activated SMADs translocate to
the nucleus and, in concert with other transcription factors, regulate gene
expression (Shi and Massague,
2003).

Here we show that activation of the TGFβ/activin/nodal branch through
SMAD2/3 is associated with pluripotency and is required for the maintenance of
the undifferentiated state in hESCs and in ex vivo mouse blastocyst
outgrowths. hESCs can be maintained undifferentiated through unknown factors
in conditioned medium or through activation of the WNT pathway by BIO, a
GSK3β inhibitor. We show that activin/nodal signaling in required
downstream of both conditions, in that inhibition of this pathway with a small
molecule inhibitor or soluble receptors results in a loss of the
undifferentiated state. Accordingly, we find that exogenous activin A is
supportive of the undifferentiated state. In our study of hESCs, we have
revealed a crucial role for TGFβ signaling in the regulation of ES cell
identity.

Cell culture

hESC line H1 was obtained from WiCell research institute and BGN1 and BGN2
lines were obtained from BresaGen. Human embryonic stem cells were maintained
as previously described (Sato et al.,
2003).

hESC synchronization

BGN1 cell cycle progression was blocked at metaphase by incubation
overnight in conditioned medium with 100 ng/ml demecolcine solution. Cells
were washed four times with PBS to release them from metaphase block and then
harvested for immunoblotting at 15 minutes and 4 hours post-release.

RT-PCR

Cells were lysed directly with 100 μl RNAbee (Tel-Test) and total RNA
was extracted. Total RNA (1 μg) was reverse transcribed to cDNA and 1/20 of
the RT reaction was used as PCR template. Radioactive amplification was
according to the following conditions: β-actin, TGGCACCACACCTTCTACAATGAGC
(forward) and GCACAGCTTCTCCTTAATGTCACGC (reverse) (21 cycles); OCT3/4,
GAAGGATGTGGTCCGAGTGT (forward) and GTGACAGAGACAGGGGGAAA (reverse) (19 cycles);
NANOG, ACCAGAACTGTGTTCTCTTCCACC (forward) GGTTGCTCCAGGTTGAATTGTTCC (reverse)
(21 cycles).

Immunofluorescence

BGN2 cells were plated on matrigel coated poly-D lysine/laminin Biocoat
coverslips (Becton-Dickinson) and cultured for 5 days in the described
conditions. Following culture, cells or mouse embryos were fixed in 4%
paraformaldehyde in PBS, washed in PBS + 0.2% bovine serum albumin (BSA),
permeabilized with 0.1% Triton X-100 in PBS/BSA for 20 minutes, and then
blocked in 5% donkey serum in PBS/BSA for 2 hours at room temperature. Cells
were incubated overnight at 4°C with combinations of either rabbit
anti-phospho-SMAD1/5 (1:100 dilution), anti-SMAD2/3 (1:500) or anti-OCT3/4
(1:500). After three 3 washes in PBS/BSA, cells were incubated with
combinations of AlexaFluor488-, AlexaFluor555- or AlexaFluor647-conjugated
donkey anti-rabbit/anti-mouse secondary antibodies for 2 hours at room
temperature. Cells were then washed with PBS/BSA and stained with either 25 nM
SytoxGreen or ToPro3-iodide nucleic acid stain in PBS/BSA. Coverslips were
mounted in glycerol and imaged using a Zeiss LSM 510 confocal microscope.

Blastocyst outgrowth

Pregnant Swiss-Webster mice were sacrificed at embryonic day 3.5 and the
uterus was isolated and flushed with warm culture medium to obtain blastocyst
stage embryos as described previously
(Nagy, 2003). Embryos were
incubated in culture medium containing 1 mg/ml pronase and observed
continuously until the zona pellucida was digested. Embryos were then rinsed
three times in warm culture medium and incubated on gelatin-coated tissue
culture plastic in mouse embryonic stem cell medium
(Betts et al., 2001) containing
either 20 μM SB431542 or an equivalent dilution of DMSO. Embryos were not
disturbed during culture in order to allow attachment and outgrowth. Embryos
were fixed in 4% paraformaldehyde in PBS at 4 days post extraction and
processed for immunofluorescence in the same manner as the hESCs. Treated
embryos and outgrowths were imaged using a Zeiss LSM 510 confocal
microscope.

Embryoid bodies formation

Embryoid body formation for BGN2 cells was carried out as previously
described (Sato et al., 2004).
BGN2 cells grown for 5 days in described conditions, harvested using dispase
(Invitrogen) and plated on bacterial culture plates on which the cells are
unable to attach. Embryoid bodies were allowed to form by growth in suspension
for 7 days. Embryoid bodies were then plated on gelatin-coated tissue culture
grade plastic and further cultured for 7 days in order to allow for terminal
differentiation of cell types. Reattached and terminally differentiated
embryoid bodies were harvested and processed by RT-PCR in order to assay for
the presence of derivatives of primary germ layers.

BrdU incorporation and TUNEL assays

The In Situ Cell Proliferation, FLUOS (cat. No. 1810740) and In Situ Cell
Death Detection, Fluorescein (cat. No. 1684795) kits were obtained from Roche.
H1 cells were cultured for 2 days in CM and then further cultured in the
indicated conditions for 3 days. Following culture, BrdU incorporation and
TUNEL staining was assayed according to the protocols described by the
manufacturer. Cells were imaged and quantified using a Zeiss LSM Pascal
confocal microscope.

Results

The undifferentiated state of hESCs is characterized by activation of
SMAD2/3 signaling and inhibition of SMAD1/5 signaling

Although TGFβ signaling has been shown to play a role in primary cell
fate decisions in many developmental models, its role in embryonic stem cell
fate determination remains uncertain. To study the involvement of the
TGFβ superfamily pathway in embryonic stem cell fate decisions, we first
analyzed the activation status of both arms of this family
(TGFβ/activin/nodal signaling and BMP/GDF signaling) in human embryonic
stem cells, both in the undifferentiated state and during the early phase of
differentiation in response to certain modulators of TGFβ superfamily
activation.

In the undifferentiated state that is maintained by growth in
MEF-conditioned medium (CM), we found that SMAD2/3 was phosphorylated and
localized to the nucleus of hESCs, indicating activation of the
TGFβ/activin/nodal pathways. Furthermore, the phosphorylation and nuclear
localization were reduced in cells that were allowed to differentiate by
growth in non-conditioned medium (nCM)
(Fig. 1A,B). We have previously
shown that BIO is also capable of maintaining hESCs in the undifferentiated
state, even in the absence of CM, through activation of canonical WNT
signaling (Sato et al., 2004;
Meijer et al., 2003).
Accordingly, BIO maintained phosphorylation of SMAD2/3 above levels seen in
hESCs grown in nCM alone, and this effect was accompanied by maintenance of
OCT3/4, a marker of pluripotency. To test the ability of hESCs cultured in nCM
to activate SMAD2/3 signaling, we cultured the cells in nCM supplemented with
activin A. Under these conditions, the cells maintained high levels of SMAD2/3
phosphorylation as well as increased OCT3/4 relative to cells cultured in nCM
alone. This suggests that SMAD2/3 activation in the absence of extrinsic
factors present in CM may be supportive of the undifferentiated state, though
not entirely sufficient to maintain typical hESC morphology nor markers of
pluripotency through extended culture (Fig.
1A-C).

The undifferentiated state of hESCs is characterized by activation of
SMAD2/3-mediated signal transduction and inhibition of SMAD1/5-mediated signal
transduction. (A) Western blot analysis of H1 hESCs cultured under various
conditions. Cells were cultured for 4 days in the presence of: nCM
supplemented with 2 μM BIO, non-conditioned medium (nCM), MEF-conditioned
medium (CM), CM supplemented with 25 ng/ml BMP4, and nCM supplemented with 25
ng/ml activin A. Membranes were probed with antibodies specific for
phosphorylated (P) SMAD2, SMAD2/3, phosphorylated SMAD1/5, SMAD1/5, OCT3/4 andα
-tubulin (as a control for protein loading). (B) Immunofluorescence
microscopy of BGN2 hESCs in the undifferentiated and differentiated state.
BGN2 cells were cultured for 5 days in CM, in nCM, in nCM supplemented with 25
ng/ml activin A, and in nCM supplemented with 2 μM BIO. Cells were
decorated with an antibody specific for SMAD2/3, as well as SytoxGreen nuclear
counterstain. (C) Bright-field images of H1 hESCs cultured in conditions
described in A. Scale bars: 50 μm.

We also examined the state of SMAD1/5 phosphorylation in hESCs grown in the
above conditions. SMAD1/5 activation in these cells showed the opposite
character of SMAD2/3 activation; in the undifferentiated state, SMAD1/5
phosphorylation was barely evident and upon differentiation, phosphorylation
was globally increased and localized to the nucleus
(Fig. 1A;
Fig. 2A). Addition of activin A
to nCM reduced phosphorylation of SMAD1/5 to levels comparable with those of
hESCs grown in nCM, demonstrating an input of SMAD2/3-mediated signaling on
the suppression of SMAD1/5 activation. To study the effect of induced SMAD1/5
activation in undifferentiated hESCs, we cultured cells in CM supplemented
with BMP4. As previously reported (Xu et
al., 2002), these conditions resulted in a decrease in OCT3/4
levels and a concomitant change in morphology
(Fig. 1A,C). Thus, we have
shown that SMAD2/3 activation correlates with stemness in hESCs descriptively
and functionally, while SMAD1/5 activation correlates with
differentiation.

Global SMAD1/5 phosphorylation is increased under differentiation
conditions and is evident in mitotic cells in the undifferentiated state. (A)
Immunofluorescence microscopy of BGN2 hESCs in the undifferentiated and
differentiated state. BGN2 cells were cultured for 5 days in CM, in nCM, in CM
supplemented with 25 ng/ml BMP4 (as a control for SMAD1/5 activation) and in
nCM supplemented with 2 μM BIO. Cells were decorated with an antibody
specific for phosphorylated SMAD1/5, as well as SytoxGreen nuclear
counterstain. Arrows indicate mitotic cells. Scale bars: 50 μm. (B) Western
blot analysis of colcemide synchronized BGN1 hESCs. Cells were blocked at
metaphase by incubation with 100 ng/ml demecolcine solution and harvested at
15 minutes and 4 hours post-release. Cells grown in CM alone and CM
supplemented with 25 ng/ml BMP4 were used as controls for asynchronous and
SMAD1/5-activated cells, respectively. Membranes were probed with antibodies
specific for phosphorylated (P) SMAD1/5, SMAD1/5, phosphorylated Ser CDKs
substrate and α-tubulin (as a control for protein loading). (C)
Immunofluorescence microscopy of blastocyst stage embryo containing mitotic
cells. Mouse blastocyst embryos were fixed and labeled with anti-phospho
SMAD1/5 antibody and SytoxGreen nuclear counterstain, and then imaged by
confocal microscopy. Scale bars: 20 μm.

Minimal global levels of SMAD1/5 phosphorylation in undifferentiated hESCs
may be accounted for by the result that phosphorylation was only evident in
the cytoplasm of cells undergoing mitosis
(Fig. 2A). In order to assess
the relationship between SMAD1/5 phosphorylation and mitotic index, hESCs were
synchronized by incubation with colcemide, which arrests cells at the onset of
metaphase. SMAD1/5 phosphorylation was compared between hESCs that had been
released from the colcemide block for different lengths of time and
asynchronous hESCs (Fig. 2B).
hESCs that were released from colcemide block for 15 minutes showed higher
levels of SMAD1/5 phosphorylation than did hESCs that had been released for 4
hours. Asynchronous hESCs show slightly reduced levels relative to cells that
had been blocked and released for 15 minutes. To measure the efficacy of
colcemide in hESC synchronization, we used an antibody that is specific for
phosphoserines on cyclin dependent kinases (CDKs) that are phosphorylated only
in the context of mitosis. BMP-stimulated hESCs were used as a control for
SMAD1/5 phosphorylation. Interestingly, a correlation between SMAD1/5
phosphorylation and mitosis was also apparent in dividing cells of
preimplantation mouse embryos (Fig.
2C).

SMAD2/3 activation is necessary for maintenance of the
undifferentiated state in hESCs

Having defined the nature of SMAD1/5 and SMAD2/3 activation in
undifferentiated and differentiating hESCs, and having discovered a
correlation between increased SMAD2/3 activation and the undifferentiated
state, we set out to determine whether active SMAD2/3 signaling is necessary
for the maintenance of the pluripotent state
(Fig. 3). The
TGFβ/activin/nodal branch of TGFβ signaling can be efficiently
inhibited by SB431542, a synthetic compound that precludes SMAD2/3
phosphorylation by type 1 TGFβ receptors
(Laping et al., 2002). We
challenged hESCs cultured in the presence of CM or BIO with SB431542 and
phosphorylation of SMAD2/3 was reduced. Furthermore, upon challenge with
SB431542, the ability of CM or BIO to maintain protein levels of the
pluripotency marker, OCT3/4, was lost. In addition to reducing SMAD2/3
phosphorylation and OCT3/4, SB431542 also has the effect of increasing SMAD1/5
phosphorylation to the levels seen in differentiating hESCs
(Fig. 3A).

Intact SMAD2/3 signaling is required for the maintenance of the
undifferentiated state in hESCs and the formation of embryoid bodies. (A)
Western blot analysis of H1 hESCs cultured in conditions in which SMAD2/3
signaling is intact or inhibited. Cells were cultured for 4 days in CM, in
nCM, in nCM supplemented with 2 μM BIO, in CM supplemented with 10 μM
SB431542, and in nCM supplemented with 2 μM BIO and 10 μM SB431542.
Membranes were probed with antibodies specific for phosphorylated (P) SMAD2,
SMAD2/3, phosphorylated SMAD1/5, SMAD1/5, OCT3/4 and α-tubulin (as a
control for protein loading). (B) Expression analysis of H1 hESCs. Cells were
cultured for 4 days in nCM supplemented with 25 ng/ml activin A, in CM
supplemented with 25 ng/ml BMP4, in CM, in nCM, in nCM supplemented with 2μ
M BIO in CM supplemented with 10 μM SB431542, and in nCM supplemented
with 2 μM BIO and 10 μM SB431542. RT-PCR was performed on these cells
using primers for human OCT3/4, nanog, β-actin (as a loading control) andβ
-actin RT minus (as a control for contamination with genomic DNA). (C)
Western blot analysis of H1 hESCs cultured in the presence of a cocktail of
soluble receptors specific to the activin/nodal pathway. Cells were cultured
for 5 days in CM, in nCM, in CM supplemented with 10 μM SB431542, and in CM
supplemented with hrActRIB (5 μg/ml), hrActRIIB (5 μg/ml) and hrCripto
(250 ng/ml). Membranes were probed with antibodies specific for phosphorylated
SMAD2, OCT3/4, NANOG and α-tubulin (as a control for protein loading).
(D) Histogram describing number of embryoid bodies formed from BGN2 hESCs.
Cells were cultured for 7 days in CM, in nCM, in nCM with 25 ng/ml activin A,
in CM with 10 μM SB431542, in nCM with 2 μM BIO, and in nCM with 2 μM
BIO and 10 μM SB431542. Cells were then detached from substrate and
cultured in a suspension of nCM for 7 more days. Histograms and error bars
(s.e.m.) represent the experiment performed in triplicate and on two separate
passages of BGN2 cells.

Prompted by the result that intact SMAD2/3 signaling was necessary for the
maintenance of high OCT3/4 protein levels in hESCs, we examined the expression
of known markers of pluripotency in hESCs with the variable levels of SMAD2/3
activation described above (Fig.
3B). Upon withdrawal of CM and the subsequent reduction of SMAD2/3
phosphorylation, hESCs show reduced expression of OCT3/4 and of NANOG, another
established marker of pluripotency. Inhibition of SMAD2/3 activation by
SB431542 in the presence of CM or BIO resulted in significantly reduced
expression of both markers. Consistently, SB431542 promoted differentiation by
morphological criteria, inducing cells to assume a flat, spread out morphology
(data not shown). Stimulation of TGFβ/activin/nodal signaling by activin
A in the presence of nCM restored the expression of both markers to levels
above those exhibited by hESCs cultured in nCM alone. Hence, by measure of two
independent markers of pluripotency, SMAD2/3 activation is not only necessary
to, but also supportive of the undifferentiated state of hESCs.

We next set out to confirm that our observations concerning the effect of
SB431542 were specific to its inhibition of TGFβ/activin/nodal signaling
through SMAD2/3, as the use of a small molecule inhibitor could potentially
affect unrelated signal transduction pathways. In order to rule out this
possibility, we used an independent means of inhibiting
TGFβ/activin/nodal signaling by culturing hESCs in CM supplemented with a
combination of human recombinant ActRIB, hrActRIIB and hrCripto
(Fig. 3C). These three proteins
form a complex and bind TGFβ/activin/nodal ligands in canonical TGFβ
signaling, so soluble extracellular domains provide an alternative to SB431542
by competing for TGFβ/activin/nodal in the CM. Both OCT3/4 and NANOG were
significantly reduced in hESCs cultured in the presence of soluble receptors
(Fig. 3C), thus providing
corroborative data demonstrating the requirement for this pathway in
pluripotent hESCs.

TGFβ signaling is involved in many of the more fundamental aspects of
cell biology, including cell viability, adhesion, migration and proliferation
(reviewed by Massague et al.,
2000). Thus, the global inhibition of ALK4/ALK5/ALK7 activation by
SB431542 could be affecting hESCs in a manner independent of differentiation.
Apart from its role in activating the TGFβ/activin/nodal branch of
TGFβ signaling, ALK4/ALK5/ALK7 has also been implicated in activation of
MAP kinases, including p38, Jnk and Erk, although the only one of these
kinases to be significantly affected by SB431542 in previously reported in
vitro assays was p38 (Inman et al.,
2002). In order to address whether SB431542 acts through means
other than inhibition of SMAD2/3 activation, we performed BrdU incorporation
and TUNEL assays to exclude the possibility that SB431542 was affecting cell
viability or proliferation (see Fig. S1A in the supplementary material) and
found no significant effect of SB431542 on these parameters. It has previously
been shown that, at the doses we have used, SB does not significantly affect
activation of other pathways in cell culture
(Inman et al., 2002). To
confirm this in hESCs, we assessed the phosphorylation of MAP kinases p38, JNK
and Erk in the presence and absence of SB431542, and found that the
phosphorylation of these effector molecules was not regulated upon addition of
SB431542 (data not shown).

Owing to the high passage number of the H1 hESC line used, it is possible
that the cells may have acquired an abnormal karyotype that endowed it with an
atypical character. In order to exclude this possibility, the cells used in
these experiments were karyotyped and found to have a normal complement of
chromosomes (data not shown).

Embryoid body formation is inhibited in SB431542 treated hESCs

Embryonic stem cells are defined functionally by their pluripotency - the
ability to give rise to cell types representing all three primary germ layers
of the embryo. In examining the necessity for SMAD2/3 signaling in the
maintenance of the pluripotency, it is important to assay the properties of
hESCs in conditions that more closely approximate in vivo conditions. There
are currently two in vivo assays for hESCs: teratoma formation in the mouse;
and formation of embryoid bodies in culture
(Thomson et al., 1998).
Embryoid bodies are formed from undifferentiated hESC aggregates cultured in
suspension and they typically contain differentiated cell types of ectodermal,
mesodermal and endodermal lineages. Differentiated hESCs do not form embryoid
bodies.

We examined the efficiency of embryoid body formation in BGN2 hESCs in
which SMAD2/3 signaling is activated or inhibited
(Fig. 3D). Addition of the
inhibitor SB431542 to cells grown in CM significantly reduced the formation of
embryoid bodies. Cells grown in the presence of BIO were able to form more
embryoid bodies than cells grown in nCM alone, and these embryoid bodies were
similar morphologically to those formed of hESCs grown in CM (data not shown).
This effect was drastically reduced in the context of SMAD2/3 inhibition and
the few embryoid bodies that did form were of an atypical, multi-cystic
morphology (data not shown). Activation of SMAD2/3 alone by activin A
conferred upon the cells a marginally increased ability to generate embryoid
bodies, which is consistent with the ability of activin A to restore OCT3/4
levels in cultured hESCs. These data support the notion that SMAD2/3
activation is necessary but only partially sufficient for maintenance of
pluripotency.

In order to assess the character of embryoid bodies formed of hESCs
cultured in the above conditions, we assayed the differentiated cell types
contained within them by RT-PCR and found that markers of all three primary
germ layers were expressed (data not shown).

SMAD2/3 activation is not necessary for maintenance of the
undifferentiated state in mESCs

Having found the same requirement for active SMAD2/3 signaling in three
independent lines of hESCs (data not shown), we next assayed the relevance of
SMAD2/3 activation to mouse embryonic stem cell identity. mESCs are typically
cultured in medium containing leukemia inhibitory factor (LIF), a protein that
has been shown to maintain mESCs (Smith,
2001), but not hESCs (Thomson
et al., 1998; Reubinoff et
al., 2000; Sato et al.,
2004), in the undifferentiated state. However, a role for LIF in
the establishment and maintenance of the stem cell compartment in vivo is
uncertain, as mouse null mutants for components of the LIF/Stat3 pathway have
no stem cell defect (Smith,
2001). We examined the nature of SMAD2/3 signaling in mESCs with
the expectation that the necessity for SMAD2/3 signaling in mESCs cultured in
medium containing LIF would be similar to that observed for hESCs cultured in
MEF conditioned medium (Fig.
4A).

Intact SMAD2/3 signaling is not required for the maintenance of the
undifferentiated state of mESCs, but is required for the maintenance of the
stem cell compartment of blastocyst outgrowths. (A) Western blot analysis of
129/SVJ mESCs. mESCs were cultured for 3 days in mESC medium with (+) and
without (-) LIF, in mESC medium with LIF plus 10 μM SB431542, in mESC
medium with 25 ng/ml activin A, in mESC medium with 2 μM BIO, in mESC
medium with 2 μM BIO plus 10 μM SB431542, in CM, and in CM with 10 μM
SB431542. Membranes were probed with antibodies specific for phosphorylated
SMAD2, SMAD2/3, OCT3/4 and α-tubulin (as a control for protein loading).
(B) Whole-mount immunofluorescent confocal microscopy of pre-implantation
stage mouse embryos. Mouse embryos were extracted and fixed at two-cell,
four-cell, eight-cell, compacted morula and blastocyst stages. Embryos were
decorated with an antibody specific for phosphorylated SMAD2 and SytoxGreen
nuclear counterstain. Scale bars: 20 μm. (C) Confocal immunofluorescent
microscopy of blastocyst outgrowths. Mouse blastocyst stage embryos were
extracted and cultured in the presence of mESC medium supplemented with DMSO
or 20 μM SB431542 for 4 days. Outgrowths were decorated with an antibody
specific for OCT3/4 and SytoxGreen nuclear counterstain. Table gives
percentage of embryos exhibiting OCT3/4-positive cells. Scale bars: 100μ
m.

mESCs were cultured in defined medium with (mESC) or without (CM) a
contribution of growth factors from serum. Upon withdrawal of LIF, levels of
SMAD2/3 phosphorylation in mESCs were reduced, but the SB431542-mediated
inhibition of SMAD2/3 signaling in the context of LIF resulted in no
significant change in OCT3/4 levels. We have previously shown that BIO is able
to maintain mESCs as well as hESCs in the undifferentiated state
(Sato et al., 2004). In
support of this result, mESCs grown in the presence of BIO were able to
maintain levels of OCT3/4 relative to mESCs cultured in the presence of LIF,
but unexpectedly, inhibition of SMAD2/3 signaling in the context of BIO had no
effect of OCT3/4 levels (Fig.
4A). Taken together, these results suggest that mESCs are
dissimilar to hESCs in that they have no requirement for active SMAD2/3
signaling in the maintenance of pluripotency. These results suggest a striking
difference between hESCs and mESCs, whereby the maintenance of the
undifferentiated phenotype requires an active SMAD2/3 signaling pathway in
hESCs but not in mESCs. Furthermore, the ability of BIO to maintain the
undifferentiated phenotype of these cells is dependent on active SMAD2/3
signaling in hESCs but not in mESCs.

Activation of SMAD2/3 is required for maintenance of pluripotency in
the ICM of mouse blastocyst outgrowths

The character of embryonic stem cells in vitro does not necessarily
recapitulate their behavior in an endogenous, in vivo context and a recent
report showing that loss of both SMAD2 and SMAD3 result in reduced epiblast
and OCT3/4 levels (Dunn et al.,
2004) prompted us to investigate the role of SMAD2/3 activation in
the mouse blastocyst. Mouse blastocyst outgrowths are used to assess the
character and potency of two cell types present in blastocyst stage embryos:
the trophoblast and inner cell mass (ICM). Ex vivo, the trophoblast cells of
the blastocyst adhere to the matrix substrates and migrate across them, while
the inner (ICM) maintains a compact morphology. It is the inner cell mass from
which pluripotent embryonic stem cells are derived, and only these cells
maintain OCT3/4 expression upon outgrowth.

To assess the requirement for SMAD2/3 activation in peri-implantation mouse
embryos, we cultured blastocyst stage embryos for 4 days in the presence or
absence of SB431542. We assayed the presence of OCT3/4 in outgrowths cultured
from the blastocyst stage for 4 days, both in the presence and absence of
SB431542 and found that 77% of control outgrowths were positive for OCT3/4
staining, while only 36% of SB431542-treated blastocysts showed OCT3/4
staining; typical control and SB431542 treated embryos in which the OCT3/4
compartment is maintained and lost, respectively, are represented in
Fig. 4C. Strikingly, although
OCT3/4 protein is present in a majority of outgrowths cultured for 4 days in
the presence of DMSO, most of those cultured in SB431542 display a complete
loss of OCT3/4 staining. Hence, SMAD2/3 activation is required not only for
maintenance of pluripotency of hESCs cultured in vitro, but it is also
required for the maintenance of the OCT3/4-positive compartment of the ICM
upon blastocyst outgrowth. Mouse blastocyst outgrowths cultured ex vivo in the
presence of the soluble receptors hrActRIB, hrActRIIB and hrCripto showed a
similar trend towards loss of the OCT3/4 positive compartment, though less
consistently than outgrowths cultured in the presence of SB431542 (data not
shown).

Discussion

For embryonic stem cells to realize their potential in clinical
applications, it is first necessary to address fundamental questions regarding
their biology and the molecular nature of `stemness'. We demonstrate here a
requirement for the TGFβ/activin/nodal branch of the TGFβ signaling
pathway in the maintenance of human embryonic stem cell identity. Either in
the context of unknown extrinsic factors secreted by MEFs (CM) or the GSK3
inhibitor BIO, inhibition of SMAD2/3 activation results in significantly
reduced expression of markers of pluripotency. Although it may contribute to
the maintenance of the undifferentiated state, SMAD2/3 activation alone does
not confer upon hESCs the stem cell identity. Indeed, WNT and
TGFβ/activin/nodal signaling collaborate in the maintenance of
pluripotency, and the WNT signaling pathway impinges, directly or indirectly,
on SMAD2/3 activation. We also found a similar requirement for SMAD2/3
signaling in the maintenance of the stem cell compartment of ex vivo mouse
blastocyst outgrowths, though activated SMAD2/3 does not seem to be necessary
for the ability of LIF to maintain the undifferentiated state of mESCs.

The primary cell fate decision of mammalian development occurs in morula
stage embryos when the outer cells of the embryo form trophoectoderm, which
mediates attachment and implantation into uterine tissue, and the inner cells
form the inner cell mass, which contributes to all the tissues of the embryo
and from which embryonic stem cells are derived (reviewed by
Rossant, 2001). Our results
indicate a role for TGFβ signaling in the maintenance of pluripotency in
the cellular derivatives of the inner cell mass. Although TGFβ
superfamily ligands have been shown to contribute to primary cell fate
determination in other vertebrate models, such as the establishment of the
dorsal organizer in Xenopus
(Harland and Gerhart, 1997), an
input for TGFβs in mammalian embryogenesis has not been well described at
pre-gastrula stages. Studies of ligand and receptor expression suggest that
the TGFβ cascade is activated in pre-implantation mouse embryos
(Albano et al., 1993;
Paria et al., 1992;
de Sousa Lopes et al., 2003)
and our analysis of SMAD2 phosphorylation at these stages showed SMAD2/3
activation as early as the four-cell stage. Yet, the role of the TGFβ
superfamily in early mammalian embryogenesis is not well understood.

Previous analyses of the role of TGFβ signal transduction in early
mammalian embryogenesis have mostly arisen from the study of knockout mice. An
array of null mutations of TGFβ signaling components have been made in
the mouse, but among these, very few have an effect before gastrulation and
none affect the establishment and/or maintenance of the stem cell compartment
at peri-implantation stages (reviewed by
Goumans and Mummery, 2000). For
example, null mutants of SMAD2 or the TGFβ/activin/nodal receptor ActRIIB
result in failure of mesoderm formation and malformed primitive streak (Song
et al., 1998). Recently, mice were created with null mutations for both SMAD2
and SMAD3 (Dunn et al., 2004).
These mice displayed a similar, though more severe, developmental phenotype to
SMAD2-null mutants, with complete failure to form mesoderm or gastrulate. A
striking character of these embryos was the loss of pluripotent epiblast by
E7.5, as measured by OCT3/4 expression, while the formation of extra-embryonic
ectoderm was retained. As extra-embryonic ectoderm arises from trophoectoderm
(Nagy et al., 2003), this double mutant phenotype supports the notion that
proper maintenance of the ICM/epiblast requires an intact SMAD2/3 signaling
pathway, because derivatives of this compartment are lost while
trophoectodermal derivatives are able to form. The double mutant phenotype
also agrees with our finding that the OCT3/4-positive compartment of
blastocyst outgrowths is lost, at the equivalent of E7.0, when SMAD2/3
signaling is globally inhibited (Fig.
4C). From these data, in combination with our results describing
the necessity for SMAD2/3 signaling in maintenance of the undifferentiated
state of hESCs, a paradigm emerges in which SMAD2/3 signaling plays a role in
the maintenance of pluripotent cell types in vivo as well as ex vivo and in
vitro.

Mice deficient for SMAD4, the common SMAD that mediates translocation of
effector SMADs to the nucleus, display a similar phenotype to the SMAD2/3
double knockout, failing to gastrulate or form mesoderm, though OCT3/4
expression in the epiblast of these embryos has not been examined
(Sirard et al., 1998). It will
be interesting to see whether markers of pluripotency are affected for this
and related phenotypes, both in vivo and upon blastocyst outgrowth. Indeed,
many of the null mutations of TGFβ superfamily ligands and receptors
should be reconsidered with respect to their effect on the epiblast.

TGFβ signaling has recently been shown by many studies to figure
prominently in the maintenance of the undifferentiated state of hESCs. Among
the factors found to be specifically expressed at high levels in
undifferentiated hESCs are Nodal
(Rosler et al., 2004),
Cripto, Lefty1 and Lefty2
(Sato et al., 2003), all
components of TGFβ signal transduction. Cripto encodes an
EGF-CFC co-receptor that is essential for responsivity to nodal, and LEFTY1
and LEFTY2 are both inhibitors of nodal signaling. Expression of nodal, LEFTY1
and LEFTY2 has been shown to be high in undifferentiated hESCs and reduced
upon differentiation (Besser,
2004), and hESCs cultured in recombinant nodal exhibit prolonged
expression of pluripotency markers
(Vallier et al., 2004).
Furthermore, TGFβ has recently been shown to contribute to a cocktail of
growth factors that maintain the undifferentiated state of hESCs in
feeder-free culture (Amit et al.,
2004) and SMAD1/5 activation by BMP4 is known to induce
trophoblast in the context of CM (Xu et
al., 2002). Our results extend the role of the TGFβ pathway
in the maintenance of the undifferentiated state of hESCs, demonstrating a
requirement for the TGFβ/activin/nodal branch downstream of canonical WNT
activation or extrinsic factors present in CM.

The correlation between SMAD1/5 phosphorylation and mitosis we have
described suggests a compelling role for ligands of the BMP/GDF branch of the
TGFβ superfamily in cell proliferation. This pathway has been linked to
cell proliferation in other developmental contexts. For example, in
Drosophila, overexpression of the BMP4 homologue dpp
promotes primordial germ cell (PGC) proliferation and causes accumulation of
more PGCs in the Drosophila gonad (Xhu and Zie, 2003). In mice, null
mutations of Alk3, the type I TGFβ receptor that mediates
SMAD1/5 activation, exhibit reduced cell proliferation in the epiblast
(Mishina et al., 2002). In
light of these phenotypes, it is not surprising to see that mitosis in two
further developmental contexts (pre-implantation mouse embryos and
undifferentiated hESCs) is coincident with SMAD1/5 phosphorylation, yet there
is no evidence to support any causal link between the two.

That SMAD2/3 signaling is not required for the LIF or BIO-mediated
maintenance of the undifferentiated state of mESCs underscores the
dissimilarity between hESCs and mESCs. LIF is sufficient to maintain
pluripotency and self-renewal of mESCs, but these cells differentiate when
cultured with LIF in the absence of serum
(Ying et al., 2003),
suggesting a necessary input from other extrinsic factors. It has recently
been shown that mESC identity can be maintained in cells cultured in the
absence of serum, as long as LIF is supplemented with either BMP4 or the
forced expression of the downstream BMP4 target, Id
(Ying et al., 2003). This
suggests an essential contribution of the TGFβ superfamily to the
maintenance of mESCs, though this contribution activates the SMAD1/5 signaling
cascade that induces differentiation to trophoblast when it is activated in
hESCs (Xu et al., 2002).
Considering the disparity between our results describing the necessity for
SMAD2/3 activation in the maintenance of pluipotency in ex vivo blastocyst
outgrowths versus mESCs, it is possible that one or both of these paradigms of
`stemness' may not recapitulate the behavior of embryonic stem cells in vivo.
Indeed, this contradiction raises questions about whether ES cells in culture
are an adequate tool for the study of embryonic development.

We have previously demonstrated the ability of BIO to maintain pluripotency
of mESCs and hESCs (Sato et al.,
2004), and our results describing the character of SMAD1/5 and
SMAD2/3 activation in hESCs extend these findings to implicate a combinatorial
role for the TGFβ and WNT signal transduction pathways in the molecular
events that underlie the undifferentiated state. In other model organisms,
namely Xenopus and zebrafish, WNT and TGFβ signaling are
believed to converge on the induction of primary cell types. In
Xenopus embryos, for example, the establishment of the organizer has
been shown to result from the dorsally localized coincidence of three events:
stabilization of β-catenin; SMAD2/3 activation by Xnr proteins; and
inhibition of SMAD1/5 activation by the BMP inhibitors chordin,
noggin and cerberus (Scialli, 2003). Input from both SMAD2/3 and
WNT signaling has been shown to be required for the expression of the BMP
inhibitory organizer genes (Xanthos et
al., 2002). Undifferentiated hESCs exhibit the same reciprocal
character with respect to SMAD activation shown here, with SMAD2/3 signaling
being active and SMAD1/5 signaling being inhibited. In light of the ability of
BIO to maintain the undifferentiated state of hESCs and the dependence of this
ability on active SMAD2/3 signaling, it is tempting to speculate that the
molecular basis of pluripotent hESC identity may be rooted in a conserved
mechanism of primary cell fate specification evident in lower vertebrates.

Embryonic stem cells are defined by their ability to self renew
indefinitely and give rise to all cell types of the embryo, yet they are
present for a relatively narrow window of the mammalian life cycle.
Maintenance of pluripotency in cultured hESCs results from the integration of
multiple signaling inputs to retain this identity through indefinite passages.
In demonstrating a requirement for TGFβ signaling, we have defined one of
the necessary inputs for the maintenance of pluripotency of hESCs. However,
the means by which SMAD2/3 activation has its effect are unclear. It remains
to be seen what targets of SMAD2/3 activation are involved in mediating the
maintenance of the undifferentiated state; and the manner in which WNT
signaling and SMAD2/3 activation collaborate to mediate pluripotency, if at
all.

Supplementary material

Acknowledgments

We thank the members of the Brivanlou laboratory for discussion and
critical reading of the manuscript. We also thank WiCell (Wisconsin) for
providing the H1 cell line, BresaGen for the BGN1 and BGN2 cell lines, and
Laurent Meijer for providing BIO. Research in A.H.B.'s laboratory, in which
this study was conducted, was supported by grants from The Rockefeller
University, the NIH (MSTP Grant GM07739 to A.J.L.) and the Juvenile Diabetes
Research Foundation (JDRFI Grant 524181).